The equilibrium state of a chemical or biological system is determined by many physical and chemical variables. Changes in one or more of these variables drive the system into a new steady state. Measurement of relaxation times provides information about the underlying properties of the system. The behavior of molecules along reaction pathways and the inter- and intra-molecular dynamics are best obtained using single molecule measurement techniques. A less explored regime involves isolation of the thermodynamic perturbation (e.g., temperature, pressure, chemical binding) on a single molecule and the subsequent observation of that same molecule. This strategy represents the ultimate sensitivity in reaction measurements because it isolates the internal degrees of freedom of a single molecule.
Over the last century, a variety of techniques have been developed to measure reaction rates in chemistry and biology. The most influential of these techniques relies on rapid mixing of reactant solutions (e.g., continuous flow/quenched flow, and stop-flow methods). In the stopped flow method, solutions containing different molecular species are driven into a mixing chamber within milliseconds, and the flow of reactants is abruptly stopped. The progress of the reaction is then monitored by following either an optical property (e.g., absorption, circular dichroism, fluorescence emission), the NMR signature of a reactant, or calorimetry. The stopped flow method has proved to be a seminal tool to probe the kinetics of enzyme activity, protein folding, proton pumping, polymerization, and drug interactions. The stopped flow method was initially limited to reactions with relatively slow time constants (t>1 s). However, variations on techniques to deliver the reactants in different ratios and the ability to mix liquids together more rapidly promise to enhance the utility of stopped flow methods and increase their bandwidths.
Other techniques were developed to study more rapid chemical and polymer kinetics. These include microfluidic and nanofluidic mixing, and relaxation methods that rapidly perturb a system from equilibrium by changes in pressure, or local chemical species concentration induced by pulses of laser light, ionic current, electrostatic potential, or mechanical force. The latter three methods allow for kinetic analysis at the nanometer length scale.
In the late 1950s, an ability to rapidly perturb solution temperature (T-jump) provided yet another means to measure what were considered at the time to be “immeasurably fast” diffusion-controlled reactions. Initial T-jump studies discharged capacitors to rapidly heat relatively large volumes of solution in microseconds. This technology was brought to the nanosecond domain with Q-switched lasers, and the temperature was estimated via a change in the optical absorbance of a tracer molecule. Infrared absorbing dyes were used to convert laser energy into heat over picosecond timescales, which enabled the study of rapid protein unfolding (e.g., RNaseA) and folding (e.g., apomyoglobin) or interfacial electron transfer reactions. Recently, an infrared laser (1445 nm) was used to directly excite an OH-stretch mode in water, leading to an increase in the temperature of picoliter volumes.
Most laser-based techniques require post processing (i.e., pump-probe, fluorescence lifetime) to deduce the local temperature changes, which limits the ability to accurately measure solution temperature in real time. In addition, each pulse from a Q-switched ultrafast laser represents an entire experiment, where the solution temperature initially increases to a predefined value and then relaxes to room temperature. A major improvement in the technique would expand the laser induced T-jump method to longer timescales in which a complex temporal profile of the temperature could be precisely controlled. This requires a much more localized heat source and an ability to measure the temperature of exceptionally small fluid volumes.